The long-term goal of this project is to elucidate the functional roles of lipid raft domains in lens fiber-cell membranes, and their associated proteins, which are crucial in maintaining the transparency of the lens. In many types of cells, lipid rafts are functionally associated with various membrane processes such as protrusions, microvilli, filopodia and lamellipodia. The lipid rafts function as platforms to recruit proteins for signal transduction, calcium transport and cholesterol transport. In lens fiber cells of all species, large numbers of interlocking domains (in the forms of ball-and-socket, protrusion and microvillus processes) protrude into neighboring cells and increase cell membrane surface area. Since the lens is a vascular organ, this unique structural configuration of interlocking domains (IDs) would be ideal for facilitating molecular exchange between cells. In this project, we focus on identification of key lipid and protein components of lipid rafts in IDs of embryonic and adult chicken lenses. Because IDs share several unique features with lipid rafts of other cells, we hypothesize that the lens-specific IDs are the lipid rafts in lens fiber cells. Aim 1 tests the prediction from this hypothesis that cholesterol is enriched in various IDs, by using filipin cytochemistry with freeze-fracture TEM. Aim 2 tests the prediction that caveolin-1, PMCA, Src kinase and other key raft-associated proteins are enriched in IDs, by using immunogold EM with high-pressure freezing procedures. Aim 3 tests the corollary of our hypothesis that cav-1-connexin interaction in ball-and- socket domains and flat cell membranes is critical for gap junction assembly and maturation in lens fibers, by using freeze-fracture immunogold replica labeling (FRIL) and biochemistry. Specifically, the roles of cav-1 in trafficking of cholesterol will be examined for the transformation of cholesterol-rich to cholesterol-free gap junctions during junction assembly, maturation, and aging using our innovative methodology combining filipin cytochemistry with FRIL at EM level. Quantitative analysis will be applied for assessing the association of cav-1 with cholesterol- containing gap junctions for their putative role as lipid-raft domains in the lens. Because the functional roles of lipid rafts critically depend on their cytoskeletal organization, Aim 4 will determine the actin branching network configuration and identify the actin-associated proteins (e.g., Arp2/3, ezrin and paralemmin) in developing raft-like domains, by using high pressure freezing procedures.